Expression of monocyte chemoattractant protein 1 in human inflamed gingival tissues.

Gingival inflammation is initiated by bacterial colonization on the tooth surface. It is characterized by infiltration of mononuclear cells, a common feature of many forms of chronic inflammation. Monocyte chemoattractant protein 1 (MCP-1) is the predominant monocyte chemoattractant secreted by a variety of different cells in vitro. For this report, we examined MCP-1 expression in bacterially induced gingival inflammation by immunohistochemistry and in situ hybridization. The cell types expressing MCP-1 are identified as vascular endothelial cells and monocytes/macrophages. Correlation analysis shows that the number of cells expressing MCP-1 is related to the degree of inflammation. Our finding that MCP-1 is expressed in inflamed gingival tissue suggests that MCP-1 plays an important role in the recruitment of monocytes and amplification of inflammatory signals in bacterially induced inflammation.     

Bacteroides-specific IgG and IgA subclass antibody-secreting cells isolated from chronically inflamed gingival tissues

The emergence of cells that produce IgG and IgA subclass antibodies to Bacteroides gingivalis (Porphyromonas gingivalis) fimbriae and lipopolysaccharide (LPS) antigens was examined in mononuclear cells isolated from inflamed gingiva of different stages (slight, moderate or advanced) of adult periodontitis (AP). Antigen-specific IgM, IgG (including IgG1, IgG2, IgG3 and IgG4) and IgA (including IgA1 and IgA2) producing cells were enumerated by the ELISPOT assay and were compared with total Ig-producing cells of each isotype or subclass. In advanced AP, the B. gingivalis fimbriae-specific IgG- and IgA-secreting cells represented 5% of total IgG- or IgA-secreting cells, while those from the moderate stage comprised approximately 1% of these two isotypes. Cells producing antibody specific for B. gingivalis LPS were observed at frequencies of 0.1% and 0.4% for IgG and IgA cells, respectively in the advanced stage. When IgG subclasses were analysed in moderate AP, the anti-fimbriae subclass responses were largely IgG1 (60%), followed by IgG2 (20%), IgG3 (10%) and IgG4 (10%). Fimbriae-specific IgG subclass responses were elevated in the advanced stage of AP, and IgG4 (40%) and IgG1 (30%) were dominant, followed by IgG3 (20%) and IgG2 (10%). IgA1 cells predominated in both the moderate and advanced stages, however a relative increase in IgA2 cells occurred in advanced AP. Mononuclear cells isolated from gingiva of AP patients did not contain cells producing antibody to antigens such as Escherichia coli K235 LPS, cholera toxin or the hapten dinitrophenyl coupled to bovine serum albumin. These results show that local IgG and IgA subclass responses occur to a protein antigen of a major periodontal disease (PD)-associated pathogen, B. gingivalis, and the increase in IgG4 and IgA2 responses may be associated with host protection.     

The characterization of lymphocytes migrating through chronically inflamed tissues.

Afferent lymphatics draining Freund's adjuvant-induced granulomas and efferent lymphatics from normal subcutaneous lymph nodes were cannulated in sheep. It was previously reported that cells collected from these lymphatics, after being radiolabelled with 111In and returned to the animal intravenously, migrated from the blood back through the granuloma or lymph node into the lymph compartment from which they were originally obtained. Afferent lymph cells preferentially migrated out of the circulation in the granuloma rather than the lymph node. The cell responsible for this selective migration was found to be a small recirculating T lymphocyte. Macrophages and lymphoblasts did not demonstrate this migration. Similarly, B cells did not contribute to the lymphocyte migration observed. The migration of lymphocytes through normal uninflamed skin was examined. Afferent cells migrated through normal skin in the same way as through a granuloma, suggesting that neither antigen nor local inflammatory changes were responsible for this migration.     

High endothelial-like venules in chronically inflamed periodontal tissues exchange polymorphs

A survey of 58 gingival biopsies revealed the presence of periodontal high endothelial-like venules (PHELVs) in chronically inflamed gingival tissues. PHELVs were found to exchange polymorphonuclear cells (PMNs) almost exclusively in advanced periodontitis, with PMNs greatly exceeding the number of mononuclear cells found in PHELVs (P less than 0.001). Electron microscopy confirmed the emigration of PMNs from these vessels. The enzyme histochemical and ultrastructural features as well as the 35SO4 uptake properties of PHELVs were similar to those of the well-characterized high endothelial venules (HEVs) of rat lymph nodes. It is generally accepted that HEVs in lymphoid tissues and inflammatory sites are specially adapted to assist in the emigration of lymphocytes. However, the observation of preferential PMN emigration in the apparent absence of lymphocyte exchange from PHELVs compels further investigation of other possible functions for HEVs. In relation to this, endothelial cells are capable of producing potent cytokines and inflammatory mediators which may contribute to the development of lesions, and the possibility is discussed that high endothelial cells are functionally adapted to enhance the production of such factors.     

Ultrastructure of chronically inflamed human submandibular glands

The structure of chronically inflamed submandibular glands from four patients was examined by electron microscopy. The patients were free of the sicca syndrome and lacked other symptoms of Sj?gren's syndrome or related autoimmune diseases. The affected glands were characterized by a reduction in acinar elements, by ductular proliferation and hyperplasia, and by large numbers of inflammatory cells in the stroma. This study indicates that many of the ultrastructural changes in salivary glands previously attributed to autoimmune diseases may also occur in patients who are free of such afflictions but whose salivary glands have undergone intermittent obstruction or infection.     

Lymphocyte isolation from chronically inflamed synovial membranes.

A simple two-step procedure was developed for isolation of lymphocytes from chronically inflamed human synovial membranes. In the first step minced inflamed synovial tissues are disrupted enzymatically by deoxyribonuclease and collagenase. The second step consists of nylon-wool column filtration of the isolated cells. 7 min of preincubation of up to 37.4 X 10(6) cells in a column packed with 600 mg nylon-wool in 6 ml prior to filtration did not result in significant selective losses of either T or B cells, whereas 45 min of preincubation did. Recovery of lymphocytes after nylon-wool column filtration ranged from 68 to 95% (mean 80%) and viability was always higher than 90%. Nylon-wool column filtration increased the proportion of lymphocytes by a mean 73%. The method allows rapid identification of synovial tissue lymphocyte subpopulations as well as characterization of their function.     

Interleukin-4 production by human alveolar macrophages

BACKGROUND: IL-4 is a key factor for T helper type 2 (Th2) differentiation and Ig class switching to IgE and IgG(4) during the development of immune responses. IL-4 is produced by T cells, mast cells, basophils, and eosinophils. However, there is also evidence suggesting that rat alveolar macrophages (AMs) produce IL-4. OBJECTIVE: Given the importance of AMs and Th2-related diseases in the lung, we investigated the production of IL-4 by human AMs. METHODS: Human AMs were isolated from bronchoalveolar lavage, purified, and IL-4 production was investigated at mRNA and protein levels using real-time PCR, flow cytometry, immunocytochemistry, and ELISA. The presence of IL-4 was investigated in subjects with asthma or asymptomatic airway hyper-responsiveness, and in normal non-smokers. RESULTS: IL-4 and IL-4delta2 (a splice variant found in other IL-4 producing cells) mRNAs were found in all these subjects, but IL-4 expression could not be correlated with a particular disease. Protein production was verified by immunocytochemistry and flow cytometry analysis demonstrating, respectively, up to 69% and 59% positive AMs, regardless of the subject condition. Furthermore, phorbol-12-myristate-13-acetate and calcium ionophore stimulated the release of IL-4 after 48 h treatment in the presence of anti-IL-4 receptor antibody. CONCLUSION: Our results show for the first time that IL-4 and IL-4delta2 mRNA are expressed and IL-4 protein produced and released by human AMs, suggesting a contribution of these cells in the modulation of Th2 immune response.     

Characteristics of mononuclear cell populations in chronically inflamed synovial membranes.

Mononuclear cells infiltrating synovial membranes in chronic synovitis were characterised both in situ and in cell suspensions by surface markers and histochemical techniques. T-lymphocytes were the predominant infiltrating cell in rheumatoid arthritis as well as in other forms of chronic arthritis, including ankylosing spondylitis and arthritis associated with Crohn's disease. B-lymphocytes were found exclusively in rheumatoid synovial membranes. These cells were demonstrable both in true germinal centres and, focally and diffusely, in nodular mononuclear infiltrates lacking the histochemical characteristics of germinal centres. The synovial lining cells, unlike mononuclear phagocytes, had no demonstrable receptors for C3 and Fc.     

Collagens synthesized in vitro by diploid fibroblasts obtained from chronically inflamed human connective tissue

Collagen synthesis by fibroblasts obtained from healthy and diseased human gingiva was compared. The cells were labeled with radioactive amino acids and the collagenous proteins synthesized were characterized after NaCl fractionation by CM-cellulose chromatography and cyanogen bromide peptide analysis. Fourteen cell lines, six from healthy gingiva, six from gingiva with chronic inflammatory periodontitis, and two from acutely inflamed gingiva were studied. All of the cell lines synthesized predominantly type I collagen. Type III collagen was a minor product of all cell lines except one from diseased tissue. Five of six cell lines from diseased gingiva and two of two from acutely inflamed tissue synthesized a collagen that was soluble in 2.5 M NaCl. The alpha1/alpha2 ratio and cyanogen bromide peptide pattern indicated that this fraction contained a collagen of the type alpha1[I]3. The alpha1[I]3 collagen was not detectable in the fibroblast lines obtained from healthy gingiva. It appears that inflamed human gingivae contain fibroblasts which differ phenotypically from cells from normal tissue in that they are capable of synthesizing alpha1[I]3 collagen.     

Locally dividing macrophages in normal and inflamed mammary glands.

Goat mammary macrophage division in vivo was assessed by detection of mitotic figures, by autoradiographic measurement of the uptake of 3H thymidine, and by a 96-well proliferation assay. Autoradiography revealed that 3.74 +/- 0.77% of nonstimulated mammary macrophages were actively synthesizing DNA. Eight days of sterile inflammation, induced by lipopolysaccharide or thioglycollate, increased mammary macrophage division (10.9 +/- 2.1%). The division increased within 2 h after inducing inflammation with thioglycollate. After 1 day, the rate of division decreased, and another increase occurred 3-4 days later. The high rate of division was maintained for greater than 60 days after the induction of sterile inflammation. Division was further shown to occur by injecting 3H-thymidine directly into the mammary gland, harvesting the macrophages 1.5 h later, and determining incorporation by autoradiography. The results of all assays of division were in agreement, suggesting they reflected the same event. The dividing cells were nonspecific esterase-positive, adherent, motile, phagocytic, and had morphological characteristics of macrophages.     

Interleukin-1 released by blood-monocyte-derived macrophages from patients with leprosy.

In highly purified blood-monocyte-derived macrophages collected from patients with leprosy and from healthy individuals and cultured in vitro with mycobacterial antigens such as Mycobacterium bovis BCG or Mycobacterium leprae, we nonspecifically induced the synthesis of interleukin-1. Normally, all supernatants from cultured macrophages of all subjects tested produced similar amounts of interleukin-1. However, only in patients with lepromatous leprosy, M. leprae, but not BCG, induced high-level synthesis of prostaglandin E2, which acted as a suppressor factor in the mouse thymocyte proliferative assay used to measure the interleukin-1 content of the supernatants. Normal interleukin-1 content of those supernatants was demonstrated by blocking the prostaglandin E2 synthesis by the addition of indomethacin to the medium throughout the experimental procedure. We also tested the efficiency of a combination of BCG and M. leprae in reducing the prostaglandin E2 synthesis, but with the methodology used, we did not observe any beneficial effect of such a combination. These results demonstrate the possible role of M. leprae in the induction of at least one of the suppressive monokines and are additional arguments for the involvement of macrophages in the suppression of the specific cell-mediated immunity to M. leprae observed in lepromatous leprosy.     

Interleukin-2 in the ontogeny of human lymphoid tissues

Cells from different fetal and adult lymphoid organs were tested for the capacity to (a) react to the T-cell growth factor interleukin-2 (IL-2) and (b) to produce IL-2 under appropriate conditions. IL-2 was determined as growth promoting activity for mouse T blasts. Optimal conditions for IL-2 production were: 5 × 10⁶ cells/ml; 4–6 µg mitogen, 24 h incubation time. Concanavalin A was preferable for fetal thymus, whereas phytohemagglutinin was the appropriate mitogen for lymphocytes from blood and lymph node. Fetal and adult spleen cells produced equal activities of IL-2 irrespective of the mitogen used. Cells from thymus and spleen of 17–21-week-old fetuses produced IL-2 activities like adult cells. Fetal liver cells from the same fetuses produced no IL-2. A comparison of IL-2 activities released from cells of 26 donors showed that within individual variations there was no decrease of the capacity to produce IL-2 with old age. An activity present in culture media of mitogen treated lymphocytes which increased thymidine uptake in fetal liver cells could be distinguished from IL-2 by its different molecular weight and by a heat treatment which abolished IL-2 but not fetal liver cell growth promoting activity. This latter activity is discussed to be colony-stimulating activity. It is concluded that IL-2 is produced by and reacts with T cells only after they have reached or passed the thymus.     

Functional and biosynthetic changes in endothelial cells of vessels in chronically inflamed tissues: evidence for endothelial control of lymphocyte entry into diseased tissues

In vitro lymphocyte adhesion to, and selective radiosulphate uptake by, endothelial cells has been demonstrated in chronically inflamed tissues of patients with peptic ulceration, rheumatoid disease, pilonidal sinus, autoimmune thyroiditis, polymyositis, primary biliary cirrhosis, and pyelonephritis. These characteristics have been described previously in endothelial cells functionally specialized for promoting lymphocyte traffic from blood to lymph node parenchyma. It is suggested that these observations indicate that some vessels in inflamed tissues may be, at least in part, responsible for the selective accumulation of lymphocytes within the tissue. Manipulating the development of this type of vessel may offer a novel way of influencing the progress of inflammatory disorders.     

P- and L-selectin mediate binding of T cells to chronically inflamed human airway endothelium

The inflammatory process that underlies allergic diseases such as asthma is characterized by tissue infiltration of eosinophils and T cells. We have used the Stamper-Woodruff frozen-section assay to characterize the receptors involved in adhesion of human peripheral blood T cells to nasal polyp endothelium (NPE) as a model of T cell migration in allergic disease. T cells bound specifically to NPE in a temperature-, cell concentration- and shear stress-dependent fashion. Adhesion was inhibited by approximately 70% by antibodies against P-selectin and its counter-receptor P-selectin glycoprotein-1 (PSGL-1). In addition, a blocking monoclonal antibody (mAb) against L-selectin caused significant although lesser inhibition. Cells adhering to NPE were primarily of the CD45RO+ memory subset. Although only a minority subset of peripheral blood T cells expressed functional PSGL-1, as determined by binding of a P-selectin Fc chimera, the majority of the P-selectin chimera-binding cells were found to be CD45RO+. This is consistent with the observation that memory T cells bind to NPE via P-selectin. Using blocking mAb we also investigated which integrins and their counter-structures were involved in T cell binding. A combination of anti-beta1 and beta2 mAb was able to inhibit adhesion by almost 50%. An antibody against intercellular adhesion molecule (ICAM)-2 gave an inhibition similar to that by anti-CD18 mAb, suggesting ICAM-2 was the major counter-receptor involved for the beta2 integrin component. This study suggests that P-selectin, and to a lesser extent L-selectin, may be acting as specific homing receptors for the airway mucosa in the context of chronic allergic disease.     

Interleukin-1 beta in human colostrum

The two forms of interleukin-1, IL-1 alpha and IL-1 beta respectively, and tumour necrosis factor (TNF) are polypeptides sharing different biological activities which are often associated with host defence mechanisms. Because of the well-recognized benefits of breast feeding for newborns, colostrum from 9 healthy lactating women was analysed for the presence of these 3 cytokines. Specific radioimmunoassay revealed that colostrum contains a significant amount of IL-1 beta (mean +/- SEM values of 1,130 +/- 259 pg/ml). The concentrations of IL-1 alpha and TNF were negligible. Colostral leukocytes are able to produce IL-1 since high activity was found after stimulation with Staphylococcus epidermidis. In addition, these cells produced IL-1 spontaneously in vitro, in contrast to resting maternal blood monocytes. As IL-1 increases resistance to infection, the presence of this cytokine represent a beneficial aspect of breast feeding.     

Interleukin-6 expression in immunologically elicited murine macrophages.

We report the expression of both interleukin-6 (IL6) messenger RNA and biological activity in complete Freund's adjuvant-elicited peritoneal macrophages (CFA-M phi). IL6 mRNA expression peaked between 4 and 8 h of lipopolysaccharide (LPS) stimulation; biological activity was maximal at approximately 18 h of stimulation. LPS-induced IL6 mRNA was inhibited by treatment with cycloheximide (5 micrograms/ml), implicating the participation of a secondary protein mediator in the induction process, or the dependence upon protein synthesis for receptor ligand interactions. Comparison of CFA-M phi with resident peritoneal macrophages suggests that the elicited cell population makes more IL6 in response to LPS than the resident population on a per cell basis.     

Characterization of the integrin and activation steps mediating human eosinophil and neutrophil adhesion to chronically inflamed airway endothelium.

We have used the Stamper-Woodruff frozen-section assay (FSA) to characterize the integrin and activation steps involved in adhesion of peripheral blood eosinophils and neutrophils to nasal polyp endothelium (NPE). Eosinophil and neutrophil adhesion was significantly inhibited by monoclonal antibodies (mAbs) against CD18 (beta2) and CD11a-c. Eosinophil adhesion was also inhibited to a lesser extent by mAbs against CD29 (beta1), CD49d (alpha4), and vascular cell adhesion molecule-1. The involvement of integrins raised the possibility of an activation step being involved in the adhesion process. Although stimulation of the cells with granulocyte macrophage colony-stimulating factor (GM-CSF) before the assay failed to modulate adhesion, binding was inhibited by up to 50% by treatment of the leukocytes with azide. In addition, neutrophil adhesion was completely abrogated by pertussis toxin (PT) and inhibited by about 50% by the platelet-activating factor antagonist WEB 2086 and antibodies against interleukin (IL)-8 and the two IL-8 receptors IL8RA and IL8RB (C-X-CR1 and -CR2). In contrast, eosinophil adhesion was unaffected by PT, WEB 2086, or anti-IL8R mAbs. mAbs against CCR-3, IL-3, IL-5, and GM-CSF also had no effect. This study demonstrates that eosinophil and neutrophil adhesion to NPE in the FSA conforms to the multistep paradigm for leukocyte adhesion and can be used to model the molecular basis for adhesion to endothelium in the context of chronic inflammatory disease. Using this assay, we have observed significant differences in integrin usage between eosinophils and neutrophils and a striking difference in the mechanism of integrin activation. These differences could explain, in part, the preferential accumulation of eosinophils in diseases such as asthma.